Human Plasma Enzyme Research Articles

Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography.

Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.

Are inactive renins in plasma and kidney related to prorenin?

The availability of elegant studies examining in vitro RNA translation of renin's message, the cDNA sequence of mouse-renin messenger RNA, and the complete amino acid sequence of active renin reveals the biosynthetic pathway of this enzyme. This pathway proceeds in a series of steps that correspond to the common mode of synthesis of secreted proteins. This information must now be correlated with a profusion of observations concerning inactive forms of renin that are found in kidney and other tissues in far higher concentration than the active enzyme in human plasma. At this time, in spite of great efforts on the part of many laboratories, the connection between the inactive renins of tissue or plasma and renin's biosynthetic precursors is unknown. Furthermore, the physiologic and pathophysiologic roles that inactive renins play, if any, have not been defined.

Studies on dopamine-converting enzymes in human plasma

Studies on dopamine-converting enzymes in human plasma

Radioimmunoassay of human plasma lecithin-cholesterol acyltransferase.

A sensitive and precise competitive-displacement double-antibody radioimmunoassay was developed for the human plasma enzyme lecithin-cholesterol acyltransferase (LCAT; Ec 2.3 1.43). The ability of plasma from various animal species to displace labeled human LCAT from goat anti-human LCAT could be ranked in the following order: man and sheep > nonhuman primates > cat or dog > pig > rabbit or guinea pig > mouse > rat. Normolipidemic subjects had levels of LCAT of 6.14 +/- 0.98 micrograms/ml (mean +/- SD, n = 66). Subjects with dysbeta-lipoproteinemia had the highest plasma LCAT levels (7.88 +/- 0.39 micrograms/ml, n = 7, P < 0.05), followed by hypercholesterolemic subjects (7.00 +/- 1.30, n = 41) and hypertriglyceridemic subjects (6.96 +/- 1.3, n = 10). LCAT-deficient subjects had the lowest enzyme levels (0.89, 0.83, and 0.05 micrograms/ml, respectively, and two subjects with no detectable enzyme). Males had lower LCAT levels (6.42 +/- 1.05 micrograms/ml, n = 90, for all subjects; 5.99 +/- 1.03, n = 44, for normolipidemics) than females (7.01 +/- 1.14, n = 34, for all subjects P < 0.01; 6.44 +/- 0.79, n = 22, for normolipidemics, P < 0.01). LCAT levels correlated significantly with total cholesterol (males, r = 0.384, P < 0.001; females, r = 0.519, P < 0.002); and total triglyceride (only in females, r = 0.512, P < 0.002). LCAT levels in females correlated inversely with HDL cholesterol (r = 0.341, P < 0.05) and apoprotein D (r = 0.443, P < 0.02), but no such relationship existed in males.

Plasma carboxypeptidase N, subunits and characteristics

Subunits of human plasma carboxypeptidase N, the enzyme that cleaves kinins and anaphylatoxins, were separated in gel filtration and in disc gel electrophoresis. During storage, the activity of the separated heavy ( M r 98,000) and light ( M r 42,000) subunits increased, while the native enzyme disappeared. Carboxypeptidase N is more stable at 37 °C in native than in dissociated form. A carboxypeptidase having about the same molecular weight as the light subunit of human plasma enzyme was purified from homogenized hog liver by using affinity chromatography. Affinity chromatography was also applied to concentrate a carboxypeptidase from hog plasma. The substrate specificity of human carboxypeptidase N was studied with 12 peptide and 1 ester substrates. Although carboxypeptidase N cleaved C-terminal lysine faster than arginine, alanine in the penultimate position increased the rate of hydrolysis of C-terminal arginine.

Substrates for kinin-releasing enzymes in human plasma.

Abstract: Indirect evidence has been provided of the presence of 3 kininogen fractions: The average amounts of kinin released by human plasma kallikrein (1.2 μg/ml plasma) and by human urine kininogenase (2.4 μg/ml plasma) when added up exceeded the amount of kinin released by combined use of the two enzyme preparations (3.0 μg/ml plasma). Methods were as described by BRISEID et al. (1968). It is suggested that plasma kallikrein released kinin from 2 kininogen fractions, SI′ and SI″, and that urine kallikrein released kinin from 2 kininogen fractions, S1″ and S2. Repeated incubation with each of the kininogenase preparations did not increase the yield of kinin. Soybean trypsin inhibitor did not reduce the amount of kinin released by urine kallikrein. In control experiments a leucine aminopeptidase preparation transformed kallidin to bradykinin, but did not increase the kinin activity of the urine kallikrein incubates. Experiments carried out . with plasma kallikrein and padutin in 60°‐heated plasma supported the assumption of the 3 kininogen fractions.

Investigations on acid phosphatase activity in human plasma and serum

Determination of acid p-nitrophenylphosphatase activity in serum or heparinized plasma gives a wrong picture of the real activity of this enzyme in human plasma. In comparison with the activity in acid-citrate-dextrose (ACD)-treated plasma, activities in serum are too high and in heparinized plasma too low. A possible explanation for the lower activities obtained in heparinized plasma is the globulin-precipitating effect of heparin under the conditions of the assay. The difference in activity of serum and ACD plasma can be attributed to acid p-nitrophenylphosphatase(s) liberated from thrombocytes during the clotting of blood. The acid p-nitrophenylphosphatase from thrombocytes, liberated during the clotting process, is completely inhibited by formaldehyde, considerably inhibited by l-tartrate and to a lesser extent by fluoride. The thrombocytic acid phosphatase also hydrolyses α-naphthyl phosphate. It is concluded, that the most reliable determination of total acid p-nitrophenylphosphatas activity as well as of “prostatic” acid p-nitrophenylphosphatase activity (tartrate-inhibited fraction) is obtained with ACD plasma.

Human plasma converting enzyme

Angiotensin-converting enzyme was partially purified from human plasma by ammonium sulfate and Sephadex gel nitration. Some of its kinetic and physiological properties were discussed in comparison with those of hog plasma-converting enzyme. The K m value for human plasma converting enzyme was obtained as 4.5 × 10 −5 m. The S 20,W value (8.2) for the enzyme was obtained by the sucrose density gradient sedimentation technique and the same molecular weight for the converting enzyme in human and hog plasma was estimated to be about 150,000 daltons. The maximum activity of the enzyme in human plasma is about 4.5 units per ml, and the calculated half-time for the conversion of angiotensin I to angiotensin II is about 7 min. This slow rate, as in hog plasma, suggests that there must be more to the “activation” of angiotensin than is immediately obvious.

Clinical pharmacologic studies of L-asparaginase.

Studies were performed with L‐asparaginase in patients with metastatic cancer and leukemia. The enzyme in human plasma had a broad optimal pH range and was stable in plasma at 40° C. for at least 40 days and at −10° C. for more than 4 months. Frequent freezing and thawing had no effect on the enzyme activity. The activity was not affected in vitro by the addition of 2 × 10−3M L‐aspartic acid, NH4C1, or (NH4)2 SO4. The initial plasma level following a single intravenous infection was dose related. The enzyme decrease in the plasma was exponential with half‐times varying among patients from 8 to 30 hours. In 15 patients who received daily doses there was a cumulative increase in plasma enzyme levels, but the half‐time did not change and rapid immune clearance was not observed. Plasma half‐time was not affected by age, sex, diagnosis, extent of disease, plasma lactic dehydrogenase levels, or hepatic or renal function. The apparent volume of distribution of the enzyme was slightly greater than the plasma volume. In a patient with a thoracic duct fistula it was found that the enzyme entered the extravascular space slowly and attained a concentration of 20 per cent the plasma level. Only a trace of L‐asparaginase activity was present in the cerebrospinal fluid and urine of patients after treatment.

Separation of amino acid naphthylamidases in human plasma on sephadex G-200 and DEAE-cellulose

Enzymatic activities for the hydrolysis of ß-naphthylamides of various amino acids (alanine, arginine, aspartic acid, glutamic acid, leucine, lysine, phenylalanine, tyrosine and valine) in human plasma were separated by column chromatography on Sephadex G-200 and DEAE-cellulose. The distribution of activities toward naphthylamides of aspartic acid and glutamic acid was identical, but different from that toward leucine naphthylamide. The distribution of activities toward naphthylamides of other aminoacids (alanine, arginine, lysine, phenylalanine, tyrosine and valine) was generally like that toward leucine naphthylamide. The molecular weight or aspartyl of glutamyl ß-naphthylamidase was 190,000, whereas that of leucyl ß-naphthylamidase was 160,000. Glutamyl ß-naphthylamidase was separated from leucyl ß-naphthylamidase by Sephadex G-200 and then DEAE-cellulose. Properties of purified ß-naphthylamidase directed toward the aspartyl or glutamyl substrates were different from those of leucyl ß-naphthylamidase. Glutamyl ß-naphthylamidase was markedly activated by CA ++, had a pH optimum at 7.5, and a K m value of 1·2 × 10 −3 M. Ca ++ had no effect on leucyl ß-naphthylamidase. Its pH optimum was 6·6 and the K m value was 3·3 × 10 −4 M. It is concluded that the enzyme in human plasma which hydrolyzes either glutamyl ß-naphthylamide or aspartyl ß-naphthylamide (aminopeptidase A) is a distinct enzyme.

Relation of the fibrinolytic mechanism to other proteolytic systems in plasma.

I shall first try to illustrate the close integration of the fibrinolytic system with other proteolytic processes in blood, by recalling some of the efforts to segregate it from the intrinsic plasma kinin forming system. Attempts at clear chemical distinction between plasmin and the specific kininogenases were complicated by two factors: (1) the activities associated with plasmin resided possibly in more than one molecular species (Markus &amp; Ambrus 1960); (2) plasma kininogenases were distinguished from plasmin only by minor quantitative or by negative criteria. The kininogenases act only on natural or synthetic substrates which are attacked by plasmin as well. The active centre of plasma kininogenase differs from that of plasmin mainly by the substrates which it does not attack. Its constitution is probably very similar to the centres of plasmin, thrombin, trypsin and chymotrypsin. Clearer differences appeared when the activation of the pre-active enzymes in human plasma was considered (Eisen 1963). Figure 1 shows that streptokinase levels activating all plasminogen in human plasma induced much smaller kinin formation than did glass powder. Yet, when clotted, the first plasma lysed in a few seconds, and the other in more than 24 hours. Similarly, the small kinin formation resulting from the complete activation by streptokinase, was greatly enhanced when activation by glass or kaolin was superimposed in the same plasma sample.

Acetaminophen Prodrugs I. Synthesis, Physicochemical Properties, and Analgesic Activity

Carbonate and carboxylic acid ester prodrugs of acetaminophen were prepared. As expected, they had lower water solubilities and higher oil/water partition coefficients than acetaminophen itself. At pH 7.4 the prodrugs were slowly hydrolyzed in phosphate buffer, but human plasma enzymes markedly accelerated the hydrolyses. The LD50's of some of the compounds in mice suggest that, following oral administration, their solubility properties play a greater part in controlling the availability of acetaminophen than do their hydrolysis rates. Several of the compounds were found to have weak analgesic activity orally in rats supporting the theory that their analgesic activities and toxicities are due to release of acetaminophen. The results show how the carbonate linkage may be employed to create prodrugs and how it is possible to obtain a series of pharmacologically similar compounds possessing vastly different physicochemical properties. Carbonate and carboxylic acid ester prodrugs of acetaminophen were prepared. As expected, they had lower water solubilities and higher oil/water partition coefficients than acetaminophen itself. At pH 7.4 the prodrugs were slowly hydrolyzed in phosphate buffer, but human plasma enzymes markedly accelerated the hydrolyses. The LD50's of some of the compounds in mice suggest that, following oral administration, their solubility properties play a greater part in controlling the availability of acetaminophen than do their hydrolysis rates. Several of the compounds were found to have weak analgesic activity orally in rats supporting the theory that their analgesic activities and toxicities are due to release of acetaminophen. The results show how the carbonate linkage may be employed to create prodrugs and how it is possible to obtain a series of pharmacologically similar compounds possessing vastly different physicochemical properties.

SOME PROPERTIES OF A CHOLESTEROL ESTERIFYING ENZYME IN HUMAN PLASMA.

Abstract A method for the radioassay of plasma cholesterol esterifying activity has been developed. Employing this method, a study has been made of the influence of pH, polyvalent cations and anions, urea, and sodium taurocholate on the cholesterol esterification reaction in human plasma. The optimal pH range is 7.5–8.5. Calcium inhibits and phosphate stimulates the reaction, but marked effects occur only in greater than physiological concentrations. The reaction is irreversibly inactivated by urea, while the inhibition by sodium taurocholate is reversible. The behavior of the cholesterol esterifying activity of human plasma on electrophoresis, DEAE-cellulose chromatography, gel filtration, ammonium sulfate precipitation, and hydroxylapatite chromatography has also been investigated; and a procedure has been developed for the preliminary purification of a cholesterol esterifying enzyme from human blood-bank plasma.

STUDIES ON PLASMA KININS

SUMMARYThe kinin‐destroying enzyme in human plasma is inhibited by metal‐binding compounds, e.g. EDTA, and also by analogues of arginine. Inhibition by EDTA appears to involve chelation of a metal on which the enzyme is dependent. The enzyme can be reactivated by a small group of metal cations (zinc, cadmium, cobalt), although other ions of similar size can be utilised to some extent. The inhibition by arginine and its analogues may be competitive. This is in keeping with the proposed carboxypeptidase nature of the enzyme.

An enzyme in human blood plasma that inactivates bradykinin and kallidins

Human plasma fraction IV-1 contains an enzyme that rapidly inactivates bradykinin and kallidin II. The activity of the enzyme was characterized with biological and chemical techniques; it inactivates bradykinin by breaking its Phe 8.Arg 9 bond.Zn 2+ or Co 2+ can reactivate the enzyme, which is inhibited by chelating agents, but Co 2+ is the only accelerator found. Numerous other compounds inhibit only. The enzyme in human red blood cells that destroys bradykinin is different from the plasma enzyme. The suggestion was made to name the enzyme in human plasma fraction IV-1 “carboxypeptidase N”.

Studies on a proteolytic enzyme in human plasma; the relationship between the proteolytic activity of plasma and blood coagulation.

A fraction of globulin was prepared from human plasma which was deficient in prothrombin, thrombin, fibrinogen, plasma thromboplastin, and accelerator globulin. The preparation of globulin contained considerable potential proteolytic activity which could be activated by streptococcal fibrinolysin. This fraction of globulin accelerated the clotting of normal platelet-deficient plasma. However, the clot-accelerating effect of the globulin fraction was the same whether or not its proteolytic property had been activated. The addition of streptococcal fibrinolysin to normal platelet-deficient plasma did not accelerate coagulation. Nor did the addition of streptococcal fibrinolysin to hemophilic platelet-deficient plasma promote its coagulation. The data presented suggest that proteolysis by activated plasma proteolytic enzyme is not an essential stage in the coagulation of the blood.

The Influence of Anesthetics on the Content of Deaminating Enzyme in Human Plasma

The Influence of Anesthetics on the Content of Deaminating Enzyme in Human Plasma